MHC-Class II restricted melanoma antigens and their use in therapeutic methods

ABSTRACT

The present invention provides MHC Class II restricted melanoma antigens recognized by CD4 +  T cells. This invention further provides prophylactic and therapeutic applications for the Class II restricted melanoma antigens. In particular, this invention provides tyrosinase Class II restricted melanoma antigens, as well as tyrosinase immunogenic peptides which have been modified to enhance their immunogenicity. These antigens can serve as an immunogens or vaccines to prevent or treat melanoma. In addition a method for isolating Class II restricted melanoma antigens or identifying new Class II restricted melanoma antigens is provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/533,895, which was filed on Sep. 26, 1995.

FIELD OF THE INVENTION

This invention is in the field of prevention and treatment of humancancers. More specifically, this invention relates to MHC Class IIrestricted melanoma antigens recognized by helper T Cells and to thepreventative, and therapeutic applications which employ these antigens.This invention also relates to methods for determining Class IIrestricted melanoma antigens.

BACKGROUND OF THE INVENTION

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells (“Cellular andMolecular Immunology” (1991) (eds) Abbas A. K., Lechtman, A. H., Pober,J. S.; W. B. Saunders Company, Philadelphia: pages 340-341). Melanomasmake up approximately three percent of all skin cancers and theworldwide increase in melanoma is unsurpassed by any other neoplasm withthe exception of lung cancer in women (“Cellular and MolecularImmunology” (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.;W. B. Saunders Company Philadelphia pages: 340-342; Kirkwood andAgarwala (1993) Principles and Practice of Oncology 7:1-16). Even whenmelanoma is apparently localized to the skin, up to 30% of the patientswill develop systemic metastasis and the majority of these will die(Kirkwood and Agarwala (1993) Principles and Practice of Oncology7:1-16). Classic modalities of treating melanoma include surgery,radiation and chemotherapy. In the past decade immunotherapy and genetherapy have emerged as new and promising methods for treating melanoma(Biologic Therapy of Cancer, 2nd ed. (1995) Devita, V. T., Hellman, S,and Rosenberg, S. A., eds; J. B. Lippincott Co., Philadelphia).

Shared melanoma-associated antigens (Ag) expressed among a variety ofmelanoma patients can be recognized by cytotoxic CD8⁺ T lymphocytesderived from melanoma patients. In short-term lysis assays, cytotoxic Tlymphocytes (CTL) grown from in vitro sensitized peripheral bloodlymphocytes (PBL) or lymph node lymphocytes, or from lymphocytesinfiltrating metastatic melanoma lesions, have been shown to recognizeautologous and MHC class I compatible allogeneic melanomas but notHLA-matched nonmelanoma tumors, lymphoblasts, or cultured fibroblasts(Darrow, D. L., Slingluff, C. L., & Siegler, H. F. (1989) J. Immunol.142, 3329-3335; Hom, S. S., et al. (1991) J. Immunother. 10, 153-164).Similar recognition patterns have been observed by measuring cytokinesecretion from tumor infiltrating lymphocytes (TIL) cocultivated withautologous or HLA-matched allogeneic tumor stimulators (Hom, S. S., etal. (1993) J. Immunother. 13, 18-30). Recently, melanoma-specific HLA-A2restricted CTL clones have been shown to recognize cultured normalmelanocytes as well as their malignant counterparts, suggesting thatshared melanoma antigens can be lineage specific (Anichini, A., et al.(1993) J. Exp. Med. 177, 989-998). To date, several class I-restrictedmelanoma-associated antigens have been molecularly defined (Van DerBruggen, P., et al. (1991) Science 254, 1643-1647; Brichard, V., et al.(1993) J. Exp. Med. 178, 489-495; Kawakami, Y., et al. (1994) Proc.Natl. Acad. Sci. USA. 91, 3515-3519; Bakker, A. B. H., et al. (1994) J.Exp. Med. 179, 1005-1009; Kawakami, Y., et al. (1994) Proc. Natl. Acad.Sci. USA. 91, 6458-6462; Gaugler B., et al. (1994) J. Exp. Med. 179,921-930). These antigens and derivative class I-restricted peptides 8 to10 amino acids in length are currently being developed as clinicalvaccines to stimulate CD8⁺ T cell responses against melanoma.

While animal models of malignant and viral diseases have shown theimportance of CD8⁺ T cells in the effector phase of the immune response,the CD4⁺ helper arm has been shown to mediate critical priming andeffector functions as well. T cell receptors on CD4⁺ T cells recognize acomplex consisting of an antigenic peptide in conjunction with an MHCClass II molecule. Unlike peptides binding to MHC class I molecules,which are restricted in length from 8-10 amino acids, the antigenicpeptides that bind Class II range from about 10 to about 34 amino acidsin length and even entire proteins. (Chicz, R. M. et al (1993) J. Exp.Med. 178, 27-47; Sette, A. et al (1989) J. Immunol 143, 1265-1267.) Thisis due to the structure of the peptide binding groove in MHC class IImolecules, which is open at both ends and allows for overhang of longerpeptides outside of the critical binding core. In contrast, the peptidebinding groove in MHC class I molecules is closed at both ends, strictlylimiting the length of possible binding peptides. (Brown, J. H. et al(1993) Nature 364, 33-39).

Strong and long lasting immunity depends in part on CD4⁺ helper T cellfunctions. Therefore the identification of Class II-restricted melanomaantigens will broaden the immunotherapeutic approaches to treatingand/or prophylaxing against melanoma.

SUMMARY OF THE INVENTION

This invention relates, in general, to MHC Class II restricted melanomaantigens recognized by CD4⁺ T-lymphocytes and the nucleic acid sequencesencoding these antigens. This invention also provides therapeutic usesfor the nucleic acid sequences, proteins or peptides described herein.In addition, this invention provides a method for identifying additionalClass II restricted melanoma antigens.

It is a general object of the present invention to provide proteins,polypeptides or peptides which encode for Class II restricted melanomaantigens.

It is another object of this invention to provide a recombinant moleculecomprising a vector and all or part of the nucleic acid sequenceencoding for a Class II restricted melanoma antigen. It is anotherobject of this invention to produce recombinant proteins or peptidesencoded by all or part of the nucleic acid sequence encoding for a ClassII restricted melanoma antigen.

It is a further object of this invention to provide methods forprophylactic or therapeutic uses for the Class II restricted melanomaantigens.

It is also an object of this invention to provide melanoma vaccinescomprising all or part of the Class II restricted melanoma antigens.

It is a further object of this invention to provide immunogenic peptidesdemonstrated to be Class II restricted melanoma antigens for use invaccines.

It is a particular object of this invention to provide tyrosinasepeptides which are Class II restricted melanoma antigens.

In addition, it is another object of this invention to providemultivalent vaccines comprising at least one Class II restrictedmelanoma antigen and at least one other immunogenic molecule capable ofeliciting an immune response in a mammal to melanoma antigens.

It is another object of this invention to provide a method forpreventing or treating melanoma utilizing Class II restricted melanomaantigens in gene therapy protocols.

It is a further object of this invention to provide peptides derivedfrom a tyrosinase protein sequence for use in vaccines.

It is yet another object of this invention to provide a method ofprophylactic or therapeutic immunization for melanoma using the vaccinesdescribed herein.

It is a further object of this invention to provide a method ofidentifying Class II restricted melanoma antigens that would constitutepotential targets for immunotherapy.

DESCRIPTION OF THE FIGURES

FIG. 1 shows that the response of CD4⁺ tumor infiltrating lymphocytes(TIL) from patient number 1088 to autologous melanoma cells is HLA-DRrestricted. TIL (1×10⁶/ml) were cultured in the presence of autologousEpstein Barr Virus (EBV)-B cells (1×10⁶/ml) alone, or B cells pulsedwith a lysate of autologous cultured melanoma cells (7×10⁵ cellequivalents/ml). Secretion of GM-CSF(granulocyte/macrophage-colony-stimulating factor) following autologoustumor stimulation was significantly inhibited by the monoclonal antibody(mAb) L243 (anti-HLA-DR) and IVA12 (anti-HLA-DR, -DP, -DQ).

FIG. 2 shows CD4⁺ TIL 1088 recognize lysates of autologous andallogeneic cultured melanoma lines presented by autologous EBV-B cells,indicating a commonly expressed melanoma antigen. TIL cultured for 58days in the presence of IL-2 were incubated for 20 hours (h) withtumor-pulsed 1088-EBV. GM-CSF secretion was measured by ELISA. All cellswere at 1×10⁶/ml.

FIG. 3 shows CD4⁺ TIL 1088 react with melanoma cells and normalmelanocytes, but not with tumors of other histologies. TIL secretedsignificant amounts of GM-CSF when stimulated with autologous EBV-Bcells pulsed with lysates of the autologous 1088-mel or normalmelanocytes FM 902 and FM 907, but not pulsed with lysates of coloncancers, breast cancers, or Ewing's sarcomas. All cells were 10⁶ per ml.Ca, cancer; Sa; sarcoma.

FIG. 4 shows that CD4⁺ TIL 1088 recognize two different tyrosinasepeptides. HLA-DRB1*0401 was identified as the presenting molecule forboth Ty 56-70 and Ty 448-462. CD4⁺TIL 1088 recognition was assessed byGM-CSF secretion. Peptides HA 307-319 and MT(65) 3-13 were used asinhibitors for B1*0401 and B1*0301, respectively.

FIG. 5 shows the MHC Class II restriction of tyrosinase peptiderecognition by CD4⁺TIL 1088 cells. The antigen presenting cells werepulsed with autologous melanoma lysate or 100 μM peptide overnight thenwashed prior to assay, all cells at 1×10⁶/ml. HLA-DR-B1*0401 wasidentified as the presenting MHC molecule for Ty 56-70 and Ty 448-462.

FIG. 6 shows in three separate experiments CD4⁺T cells from patient 1088showed specific recognition of the Ty 56-70 peptide. Mutated andtruncated peptides were used to identify the primary anchor (binding)residues within Ty 56-70. Recognition was measured as GM-CSF secretionby T cells cocultured for 24 hours with peptide-pulsed autologousEBV-transformed B cells. Sequence identifiers for FIG. 6: Ty 56-70, SEQID NO: 1; Ty 56-70 I58→Q, SEQ ID NO: 17; Ty 56-70 I58→F, SEQ ID NO: 18;Ty 56-70 I58→V, SEQ ID NO: 19; Ty 56-70 L59→Q, SEQ ID NO: 20; Ty 56-60L59→F, SEQ ID NO: 21; Ty 56-70 L59→V. SEQ ID NO: 22 Ty 56-70 L60→Q, SEQID NO: 23 Ty 56-70 L60→F, SEQ ID NO: 24; Ty 56-70 L60→V, SEQ ID NO: 25;Ty 56-70 A63→Q, SEQ ID NO: 26; Ty 56-70 A63→V. SEQ ID NO: 5; Ty 56-70P64→Q, SEQ ID NO: 27; Ty 56-70 P64→V, SEQ ID NO: 28; Ty 56-70 L65→Q, SEQID NO: 29; Ty 56-70 L65→V, SEQ ID NO: 3; Ty 57-70, SEQ ID NO: 2; Ty58-70, SEQ ID NO: 30; Ty 59-71, SEQ ID NO: 31; Ty 60-7, SEQ ID NO: 32;Ty 61-75, SEQ ID NO: 33.

FIG. 7 shows the tyrosinase (Ty) peptides Ty 56-70 and Ty 448-462 withthe P1 and P6 anchor positions boxed. the number designation of thepeptide (e.g. Ty 56-70) indicates the amino acids spanned by thepeptide, with 1 being the methionine encoded by the intiation codon.

FIG. 8 shows CD4⁺1088 TIL recognize modified tyrosinase 56-70 (Ty 56-70)peptides better than the nonmutated or non-modified peptide. (BackgroundTIL+EBV=118 pg/ml, subtracted.)

FIG. 9 shows in two experiments, Ty 448-462 was specifically recognizedby CD4⁺T cells from patient 1088. Mutated and truncated peptides wereused to identify Y451 as the primary P1 binding residue within Ty448-452. Sequence identifiers for FIG. 9: Ty 448-462, SEQ ID NO: 6; Ty448-462 Y449→Q, SEQ ID NO: 8; Ty 448-462 Y449→F, SEQ ID NO: 9; Ty448-462 Y451→Q, SEQ ID NO: 34; Ty 448-462 Y451→F, SEQ ID NO: 10; Ty448-462 L452→Q, SEQ ID NO: 35; Ty 448-462 L452→F, SEQ ID NO: 36; Ty449-462, SEQ ID NO: 13; Ty 450-462,SEQ ID NO: 14; Ty 451-462, SEQ ID NO:37; Ty 452-462, SEQ ID NO: 38.

FIG. 10 shows CD4⁺ TIL 1088 recognize truncated tyrosinase 448-462peptides, Ty 449-462 and Ty 450-462, better than Ty 448-462. (BackgroundTIL+EBV=196 pg/ml, subtracted).

FIG. 11 shows the P6 anchor position for Ty 448-462 is D456. This waspresumed, based on identifying Y451 as the P1 anchor. D is not anoptimal residue in this position, and a valine substitution led tomarkedly enhanced CD4⁺ T cell recognition. When two favorablemodifications of Ty 448-462 were combined in a single modified peptide(Ty 450-462, D456 V), recognition was enhanced even more (see FIG. 12).

FIG. 12 shows CD4⁺ TIL 1088 recognize modified Tyrosinase 448-462peptides better than the non-modified or nonmutated peptide (BackgroundTIL+EBV=99 pg/ml, subtracted).

DETAILED DESCRIPTION OF THE INVENTION

Major Histocompatibility Complex (MHC) is a generic designation meant toencompass the histo-compatibility antigen systems described in differentspecies, including the human leucocyte antigens (HLA).

The term melanoma includes, but is not limited to, melanomas, metastaticmelanomas, melanomas derived from either melanocytes or melanocyterelated nevus cells, melanocarcinomas, melanoepitheliomas,melanosarcomas, melanoma in situ, superficial spreading melanoma,nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma,invasive melanoma or familial atypical mole and melanoma (FAM-M)syndrome. Such melanomas in mammals may be caused by, chromosomalabnormalities, degenerative growth and developmental disorders,mitogenic agents, ultraviolet radiation (UV), viral infections,inappropriate tissue expression of a gene, alterations in expression ofa gene, and presentation on a cell, or carcinogenic agents. Theaforementioned melanomas can be diagnosed, assessed or treated bymethods described in the present application.

Immunogenic peptide includes, but is not limited to, an antigenicpeptide capable of causing or stimulating a cellular or humoral immuneresponse. Such peptides may also be reactive with antibodies.

This invention provides MHC-Class II restricted melanoma antigens. Suchantigens may be the complete protein encoded by gene, or portionsthereof or polypeptides or peptides derived from a protein sequence.Such antigens may be expressed in normal or disease tissues. By way ofexample, Class II restricted melanoma antigens may be derived from thetyrosinase amino acid sequences. Examples of immunogenic tyrosinasesequences that may be used include, but are not limited to, GenBankaccession numbers J03581, U01873, Y00819, and M27160, (Kwon, et al.,(1987) PNAS 84:7473-7477; Brichard, V. et al., (1993) J. Exp. Med.178:489-495; Bouchard, B. et al. (1989) J. Exp. Med. 169:2029-2042; andShibihara, S. et al., (1988) J. Exp. Med. 156:403-414; all hereinincorporated by reference). The Class II restricted melanoma antigen maycomprise the entire tyrosinase sequence or portions thereof. Examples ofimmunogenic tyrosinase peptides recognized by CD4⁺T cells include, butare not limited to, QNILLSNAPLGPQFP (Ty 56-70) (SEQ ID NO:1),NILLSNAPLGPQFP (Ty 57-70) (SEQ ED NO:2), DYSYLQDSDPDSFQD (Ty 448-462)(SEQ ID NO:6), YSYLQDSDPDSFQD (Ty 449-462) (SEQ ID NO: 13), andSYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14) (Peptides are presented insingle letter code). Also intended to be encompassed by this inventionare proteins or polypetides comprising these immunogenic peptidesequences. Persons of ordinary skill in the art will recognize thatthese peptides could be shortened to a minima MHC Class II binding coreor 9 or 10 amino acids by truncating the amino and/or carboxy termini ofthese peptides or one could lengthen these peptides by adding flankingsequences at either the carboxy or amino terminus of the peptides or atboth termini of the peptides. By way of example, such a peptide mayrange in size from about 9 amino acids to about 34 amino acids.

This invention further includes analogs of these immunogenic peptidesderived from the tyrosinase amino acid sequence. The term analogincludes any peptide which displays the functional aspects of theseimmunogenic peptides. The term analog also includes conservativesubstitution or chemical derivative of the peptides as described above.These peptides may be synthetically or recombinantly produced byconventional methodology.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to the sequences described herein inwhich one or more residues have been conservatively substituted with afunctionally similar residue and which displays the functional aspectsof the peptides as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those proteins or peptideswhich contain one or more naturally-occurring amino acid derivatives ofthe twenty standard amino acids. For examples: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Proteins, polypeptides or proteins of the present invention also includeany polypeptide having one or more additions and/or deletions orresidues relative to the sequence of a polypeptide or peptide whosesequence are described herein, so long as the requisite activity ismaintained.

In yet another embodiment of this invention, Class II restrictedpeptides derived from a tyrosinase sequence are modified to increaseimmunogenicity by enhancing the binding of the peptide to the MHC ClassII molecule with which the peptide is associated when presented to CD4⁺Tcells, or by enhancing binding of the peptide to the T cell receptor ofthe CD4⁺T cells. By way of example, modifications may include thesubstitution, deletion or addition, of one or more amino acids withinthe peptide sequence, or insertion of amino acids within the givenpeptide sequence or derivitization of existing amino acids within thegiven peptide sequence or mutation of the amino acids within the givenpeptide sequence. Examples of modified QNILLSNAPLGPQFP (Ty 56-70) (SEQID NO:1) peptide include, but are not limited to, QNILLSNAPVGPQFP(L65→V), (SEQ ID NO:3) QNILLSNVPVGPQFP (A63→V and L65→V) (SEQ ID NO:4),and QNILLSNVPLGPQFP (A63→V) (SEQ ID NO:5) (See FIG. 6). Examples ofmodified DYSYLQDSDPDSFQD (Ty 448-462) (SEQ ID NO:6) peptides include,but are not limited to DYSYLQDSDPDSSQD (F460→S) (SEQ ID NO:7),DQSYLQDSDPDSFQD (Y449→Q) (SEQ ID NO:8), DFSYLQDSDPDSFQD (Y449→F) (SEQ IDNO:9), DYSFLQDSDPDSFQD (Y451→F) (SEQ ID NO:10), DYSYLQDSVPDSFQD (D456→V)(SEQ ID NO: 11), and SYLQDSVPDSFQD (Ty450-462, D456→V) (SEQ ID NO:12(see FIGS. 9 and 11).

Preferably the modifications are performed within the Class II corebinding of the tyrosinase peptides. In a preferred modification at leastone amino acid is substituted or replaced in the given binding core ofthe immunogenic peptide sequence. Any amino acid composing the givenbinding core of the immunogenic peptide sequence may be modified inaccordance with this invention. Any amino acid may be used to substituteor replace a given amino acid within the binding core of the immunogenicpeptide sequence. Modification may occur at any amino acid positionwithin the binding core of an immunogenic tyrosinase peptide. Modifiedpeptides is intended to include any modified peptide exhibiting enhancedbinding with the MHC Class II molecule with which it is associated whenpresented to the CD4⁺ T cell. Also intended to be encompassed by thisinvention are proteins or polypeptides comprising or including thesepeptide sequences. By way of example such proteins or polypeptides mayhave additional sequences such as flanking sequences either at thecarboxy or amino terminus of the peptide or both.

By way of example, the Class II restricted tyrosinase antigens may berecognized by CD4⁺T cells in the context of HLA-DR, in particularHLA-DRB1*0401. The core binding sequence of a Class II restrictedantigen is about 9 amino acids in length. Preferably for enhancedbinding of the peptide to HLA-DRB1*0401 the first position in the coreamino acid sequence is an aromatic or aliphatic hydrophobic amino acid.The sixth position may be any hydrophobic amino acid such as, but notlimited to, leucine, isoleucine, valine, methionine, or a hydroxyl aminoacid, such as serine or threonine (Sette, A. et al (1993) J. Immunol.151, 3163-3170; Rammensee, H. G. et al (1995) Immunogenetics 41:178-228,both herein incorporated by reference).

The fourth, seventh and ninth positions of the 9 amino acid binding coresequence of the immunogenic peptide may also be substituted or replaced.Examples of amino acids that may be used at the fourth position of thepeptide include, but are not limited to, any hydrophobic amino acid oraspartic or glutamic acid. The seventh position may be any polar,charged or aliphatic amino acid. Examples of amino acids that may beused include but are not limited to aspartic acid, alanine, serine,valine, histidine, proline, asparagine, methionine, threonine, leucineand isoleucine. The ninth position of the peptide may be any polar oraliphatic amino acid. Examples of such amino acids include but are notlimited to alanine, serine, glutamine, glycine, leucine, valine, andthreonine.

Examples of Class II restricted tyrosinase peptides whose core sequencemay be modified in accordance with the present embodiment include, butis not limited to QNILLSNAPLGPQFP (Ty 56-70) (SEQ ID NO: 1),NILLSNAPLGPQFP (Ty 57-70) (SEQ ID NO: 2), DYSYLQDSDPDSFQD (Ty 448-462)(SEQ ID NO: 6), YSYLQDSDPDSFQD (Ty 449-462) (SEQ ID NO: 13), andSYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14). Examples of modifiedtyrosinase peptides whose core binding sequence may further be modifiedinclude, but is not limited to, QNILLSNAPVGPQFP (Ty56-70, L65→V) (SEQ IDNO:3), QNILLSNVPVGPQFP (Ty 56-70, A63→V and L65→V) (SEQ ID NO: 4),QNILLSNVPLGPQFP (Ty 56-70, A63→V) (SEQ ID NO: 5), DYSYLQDSDPDSSQD (Ty448-462, F460→S) (SEQ ID NO: 7), DQSYLQDSDPDSFQD (Ty 448-462, Y449→Q)(SEQ ID NO: 8), DFSYLQDSDPDSFQD (Ty 448-462, Y449→F) (SEQ ID NO: 9),DYSFLQDSDPDSFQD (Ty 448-462, Y451→F) (SEQ ID NO: 10), DYSYLQDSVPDSFQD(Ty 448-462, D456→V) (SEQ ID NO: 11), and SYLQDSVPDSFQD (Ty450-462,D456→V) (SEQ ID NO: 12).

By way of example modified Class IL-restricted tyrosinase peptidesderived from the tyrosinase sequence may have a binding core sequenceaccording to the formula X₁LLX₂NX₃X₄LX₅ (SEQ ID NO: 15), orX₁LQX₂SX₃X₄DX₅ (SEQ ID NO: 16), wherein:

X₁ may be any hydrophobic amino acid, either aromatic or aliphatic.Examples of amino acids that may be used include, but are not limitedto, leucine, isoleucine, methionine, valine, tryptophan, phenylalanine,or tyrosine. The X₁ position corresponds to Ty 58 in peptide Ty56-70, orTy 451 in peptide Ty 448-462.

X₂ may be any hydrophobic amino acid, or aspartic acid or glutamic acid.Examples of amino acids that may be used include, but are not limitedto, phenylalanine, tryptophan, leucine, isoleucine, valine, alanine,aspartic acid or glutamic acid.

X₃ may be any hydrophobic amino acid, or hydroxylamino acids. Examplesof amino acids that may be used include, but are not limited to,leucine, isoleucine, methionine, valine, serine or threonine.

X₄ may be any polar, charged or aliphatic amino acid. Examples of aminoacids that may be used include, but are not limited to, aspartic acid,alanine, serine, valine, histidine, proline, asparagine, methionine,threonine, leucine, and isoleucine.

X₅ may be any polar, or aliphatic amino acid. By way of example aminoacids that may be used on this position include, but are not limited to,alanine, serine, glutamine, glycine, leucine, valine, and threonine.

This invention further includes analogs of these modified peptidesderived from the tyrosinase sequence. The term analog is intended toinclude any peptide which displays the functional aspects of thesemodified peptides as described above. These modified peptides may besynthetically or recombinantly produced by conventional methods. Alsointended to be encompassed by this invention are proteins orpolypeptides including these peptide sequences. By way of example suchproteins or polypeptides may have additional flanking sequences ateither the carboxy or amino terminus of the peptide or at both terminior may be truncated to a minimal 9 amino acid MHC Class II binding core.

This invention also relates to nucleic acid encoding the Class IIrestricted immunogenic tyrosinase peptides or modified peptides of thisinvention. It is, however, understood by one skilled in the art that dueto the degeneracy of the genetic code variations in nucleic acidsequences will still result in a DNA sequence capable of encodingantigens described herein. Such DNA sequences are therefore functionallyequivalent to the sequences intended to be encompassed by the invention.Allelic variations in a given species of the nucleic acid sequenceencompassed by this invention are also intended to be encompassed by thepresent invention. By way of example nucleic acid sequences encoding thetyrosinase or modified tyrosinase peptides QNILLSNAPLGPQFP (Ty 56-70)(SEQ ID NO: 1), NILLSNAPLGPQFP (Ty 57-70) (SEQ ID NO: 2),DYSYLQDSDPDSFQD (Ty 448-462) (SEQ ID NO: 6), YSYLQDSDPDSFQD (Ty 449-462)(SEQ ID NO: 13), SYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14),QNILLSNAPVGPQFP (Ty 56-70, L65→V) (SEQ ID NO: 3), QNILLSNVPVGPQFP (Ty56-70, A63→V and L65→V) (SEQ ID NO: 4), QNILLSNVPLGPQFP (Ty 56-70,A63→V) (SEQ ID NO: 5) DYSYLQDSDPDSSQD (Ty 448-462, F460→S) (SEQ ID NO:7), DQSYLQDSDPDSFQD (Ty 448-462, Y449→Q) (SEQ ID NO: 8), DFSYLQDSDPDSFQD(Ty 448-462, Y449→F) (SEQ ID NO: 9) DYSFLQDSDPDSFQD (Ty 448-462, Y451→F)(SEQ ID NO: 10), DYSYLQDSVPDSFQD (Ty-448-462, D456→V) (SEQ ID NO: 11),and SYLQDSVPDSFQD (Ty450-462, D456→V) (SEQ ID NO: 12) or analogs thereofare intended to be encompassed by this invention.

This invention also provides a recombinant DNA molecule comprising allor part of the nucleic acid sequence encoding a Class II melanomaantigen and a vector. Expression vectors suitable for use in the presentinvention comprise at least-one expression control element operationallylinked to the nucleic acid sequence. The expression control elements areinserted in the vector to control and regulate the expression of thenucleic acid sequence. Examples of expression control elements include,but are not limited to, lac system, operator and promoter regions ofphage lambda, yeast promoters and promoters derived from polyoma,adenovirus, retrovirus or SV40. Additional preferred or requiredoperational elements include, but are not limited to, leader sequence,termination codons, polyadenylation signals and any other sequencesnecessary or preferred for the appropriate transcription and subsequenttranslation of the nucleic acid sequence in the host system. It will beunderstood by one skilled in the art that the correct combination ofrequired or preferred expression control elements will depend on thehost system chosen. It will further be understood that the expressionvector should contain additional elements necessary for the transfer andsubsequent replication of the expression vector containing the nucleicacid sequence in the host system. Examples of such elements include, butare not limited to, origins of replication and selectable markers. Itwill further be understood by one skilled in the art that such vectorsare easily constructed using conventional methods (Ausubel et al.,(1987) in “Current Protocols in Molecular Biology”, John Wiley and Sons,New York, N.Y.) or are commercially available.

Another aspect of this invention relates to a host organism into which arecombinant expression vector containing all or part of the nucleic acidsequence encoding for a Class II melanoma antigens has been inserted.The host cells transformed with the nucleic acid sequences encompassedby this invention include eukaryotes, such as animal, plant, insect andyeast cells and prokaryotes, such as E. coli. The means by which thevector carrying the gene may be introduced into the cell include, butare not limited to, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art (Sambrook et al. (1989) in“Molecular Cloning. A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.).

In a preferred embodiment, eukaryotic expression vectors that functionin eukaryotic cells are used. Examples of such vectors include, but arenot limited to, retroviral vectors, vaccinia virus vectors, adenovirusvectors, herpes virus vector, fowl pox virus vector, plasmids, such aspCDNA3 (Invitrogen, San Diego, Calif.) or the baculovirus transfervectors. Preferred eukaryotic cell lines include, but are not limitedto, COS cells, CHO cells, HeLa cells, NIH/3T3 cells, 293 cells (ATCC#CRL1573), T2 cells, dendritic cells, monocytes or Epstein-Barr Virustransformed B cells. In a preferred embodiment the recombinant proteinexpression vector is introduced into mammalian cells, such as NIH/3T3,COS-7, CHO, 293 cells (ATCC #CRL 1573), T2 cells, dendritic cells, ormonocytes to ensure proper processing and modification of the protein.

The recombinant protein expressed by the host cells can be obtained as acrude lysate or can be purified by standard protein purificationprocedures known in the art which may include differentialprecipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. (Ausubel et. al., (1987) in“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N.Y.). In the case of immunoaffinity chromatography, the recombinantprotein may be purified by passage through a column containing a resinwhich has bound thereto antibodies specific for the tyrosinase protein(Ausubel et. al., (1987) in “Current Protocols in Molecular Biology”John Wiley and Sons, New York, N.Y.).

This invention further includes an antibody or antibodies reactive withthe Class II restricted melanoma antigens described. The antibodies maybe monoclonal and polyclonal and are made by conventional methods knownto those skilled in the art. In addition, the protein or nucleic acidsequences of the Class II restricted melanoma antigens described herein,may be used diagnostically to screen for the presence, absence oralteration in expression of these antigens using immunoassays or nucleicacid probes.

The Class II restricted melanoma antigens of this invention, or analogsthereof may be used as a vaccine either prophylactically ortherapeutically. When provided prophylactically the vaccine is providedin advance of any evidence of melanoma. The prophylactic administrationof the Class II restricted melanoma antigen vaccine should serve toprevent or attenuate melanoma in a mammal. In a preferred embodimentmammals, preferably human, at high risk for melanoma areprophylactically treated with the vaccines of this invention. Examplesof such mammals include, but are not limited to, humans with a familyhistory of melanoma, humans with a history of atypical moles, humanswith a history of FAM-M syndrome or humans afflicted with melanomapreviously resected and therefore at risk for reoccurrence. Whenprovided therapeutically, the vaccine is provided to enhance thepatient's own immune response to the tumor antigen present on themelanoma or metastatic melanoma. The vaccine, which acts as animmunogen, may be a cell, cell lysate from cells transfected with arecombinant expression vector, cell lysates from cells transfected witha recombinant expression vector encoding for the Class II restrictedmelanoma antigen, or a culture supernatant containing the expressedprotein. Alternatively, the immunogen is a partially or substantiallypurified recombinant protein, peptide or analog thereof encoding for aClass II restricted melanoma antigen. The proteins or peptides may beconjugated with lipoprotein or administered in liposomal form or withadjuvant using conventional methodologies. Examples of Class IIrestricted tyrosinase peptides or modified tyrosinase peptides that maybe used include, but are not limited to, QNILLSNAPLGPQFP (Ty 56-70) (SEQID NO: 1), NLLSNAPLGPQFP (Ty 57-70) (SEQ ID NO: 2), DYSYLQDSDPDSFQD (Ty448-462) (SEQ ED NO: 6), YSYLQDSDPDSFQD (Ty 449-462) (SEQ ID NO: 13),SYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14), QNILLSNAPVGPQFP (Ty56-70,L65→V) (SEQ ID NO: 3), QNILLSNVPVGPQFP (Ty 56-70, A63→V and L65→V) (SEQID NO: 4), QNILLSNVPLGPQFP (Ty 56-70, A63→V) (SEQ ID NO: 5),DYSYLQDSDPDSSQD (Ty 448-462, F460→S) (SEQ ID NO: 7), DQSYLQDSDPDSFQD (Ty448-462, Y449→Q) (SEQ ID NO: 8), DFSYLQDSDPDSFQD (Ty 448-462, Y449→F)(SEQ ID NO: 9), DYSFLQDSDPDSFQD (Ty 448-462, Y451→F) (SEQ ID NO: 10),DYSYLQDSVPDSFQD (Ty 448-462, D456→V) (SEQ ID NO: 11), and SYLQDSVPDSFQD(Ty450-462, D456→V) (SEQ ID NO: 12) or analogs thereof. The tyrosinaseprotein or tyrosinase peptides having the modified binding coresequences described herein may also be used.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation.

The formulations of the present invention, both for veterinary and forhuman use, comprise an immunogen as described above, together with oneor more pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of about 0.11 about 10,000 parts by weight perpart by weight of immunogen. If two or more stabilizers are to be used,their total amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of about 0.1 to about 3.0 osmoles,preferably in the range of about 0.8 to about 1.2. The pH of the aqueoussolution is adjusted to be within the range of about 5.0 to about 9.0,preferably within the range of 6-8. In formulating the immunogen of thepresent invention, anti-adsorption agent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate thetyrosinase protein, peptides and analogs thereof into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of akit, alone, or in the form of a pharmaceutical composition as describedabove.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic or enhance theprotein's immunogenecity. Examples of such carrier molecules include butare not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin(KLH), tetanus toxoid, and the like. The immunogen also may be coupledwith lipoproteins or administered in liposomal form or with adjuvants.The immunogen can be administered by any route appropriate for antibodyproduction such as intravenous, intraperitoneal, intramuscular,subcutaneous, and the like. The immunogen may be administered once or atperiodic intervals until a significant titer of CD4⁺ or CD8⁺ T cell orantibodies directed against the Class II restricted melanoma antigen isobtained. The presence of cells may be assessed by measuring cytokinesecretion in response to antigen-presenting cells pulsed with theimmunogen. The antibody may be detected in the serum using conventionalimmunoassays.

The administration of the vaccine or immunogen of the present inventionmay be for either a prophylactic or therapeutic purpose. When providedprophylactically, the immunogen is provided in advance of any evidenceor in advance of any symptom due to melanoma, or in patients renderedfree of disease by conventional therapies but at significant risk forrecurrence. The prophylactic administration of the immunogen serves toprevent or attenuate melanoma in a mammal. When providedtherapeutically, the immunogen is provided at (or after) the onset ofthe disease or at the onset of any symptom of the disease. Thetherapeutic administration of the immunogen serves to attenuate thedisease.

By way of example, a vaccine prepared using recombinant expressionvectors may be used. To provide a vaccine to an individual a geneticsequence which encodes for all or part of the Class II restrictedmelanoma antigen is inserted into an expression vector, as describedabove, and introduced into the mammal to be immunized. Examples ofvectors that may be used in the aforementioned vaccines include, but arenot limited to, defective retroviral vectors, adenoviral vectors,vaccinia viral vectors, fowl pox viral vectors, or other viral vectors(Mulligan, R. C., (1993) Science 260:926-932). The viral vectorscarrying the nucleic sequence can be introduced into a mammal eitherprior to any evidence of melanoma or to mediate regression of thedisease in a mammal afflicted with melanoma. Examples of methods foradministering the viral vector into the mammals include, but are notlimited to, exposure of cells to the virus ex vivo, or injection of theretrovirus or a producer cell line of the virus into the affected tissueor intravenous administration of the virus. Alternatively the viralvector carrying all or part of the tyrosinase nucleic acid sequenceencoding the Class II restricted melanoma antigen may be administeredlocally by direct injection into the melanoma lesion or topicalapplication in a pharmaceutically acceptable carrier. Examples ofnucleic acid sequences that may be used include, but are not limited to,nucleic acid sequence encoding the Class II tyrosinase restrictedpeptides or modified peptides QNILLSNAPLGPQFP (Ty 56-70) (SEQ ID NO: 1),NILLSNAPLGPQFP (Ty 57-70) (SEQ ID NO: 2), DYSYLQDSDPDSFQD (Ty 448-462)(SEQ ID NO: 6), YSYLQDSDPDSFQD (Ty 449-462) (SEQ ID NO: 13),SYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14), QNILLSNAPVGPQFP (Ty 56-70,L65→V) (SEQ ID NO: 3), QNILLSNVPVGPQFP (Ty 56-70, A63→V and L65→V) (SEQID NO: 4), QNILLSNVPLGPQFP (Ty 56-70, A63→V) (SEQ ID NO: 5),DYSYLQDSDPDSSQD (Ty 448-462, F460→S) (SEQ ID NO: 7), DQSYLQDSDPDSFQD (Ty448-462, Y449→Q) (SEQ ID NO: 8), DFSYLQDSDPDSFQD (Ty 448-462, Y449→F)(SEQ ID NO: 9), DYSFLQDSDPDSFQD (Ty 448-462, Y451→F) (SEQ ID NO: 10),DYSYLQDSVPDSFQD (Ty 448-462, D456→V) (SEQ ID NO: 11), and SYLQDSVPDSFQD(Ty450-462, D456→V) (SEQ ID NO: 12) or analogs thereof. In addition,nucleic acid sequences encoding tyrosinase peptides comprising themodified core binding sequences described herein may also beincorporated into recombinant vectors. The quantity of viral vector,carrying the nucleic acid sequence encoding for the Class II restrictedmelanoma antigen, to be administered is based on the titer of virusparticles. By way of example, a range of the immunogen to beadministered may be about 10⁶ to about 10¹¹ virus particles per mammal,preferably a human. After immunization the efficacy of the vaccine canbe assessed by production of antibodies or immune cells that recognizethe antigen, as assessed by specific cytokine production or by tumorregression. One skilled in the art would know the conventional methodsto assess the aforementioned parameters. If the mammal to be immunizedis already afflicted with melanoma or metastatic melanoma the vaccinecan be administered in conjunction with other therapeutic treatments.Examples of other therapeutic treatment includes, but are not limitedto, adoptive T cell immunotherapy, coadministration of cytokines orother therapeutic drugs for melanoma.

Alternatively all or parts thereof of a substantially or partiallypurified tyrosinase protein corresponding to the Class II restrictedmelanoma antigen or polypeptides or peptides may be administered as avaccine in a pharmaceutically acceptable carrier. By way of example,ranges of protein polypeptides or peptides to be administered may be0.001 to about 100 mg per patient, preferred doses are about 0.01 toabout 10 mg per patient. In a preferred embodiment, tyrosinase Class IIrestricted peptide melanoma antigens or analogs thereof or modifiedtyrosinase peptides are administered therapeutically or prophylacticallyto a mammal in need of such treatment. By way of example, doses may beabout 0.001 mg to about 100 mg, preferred doses are about 0.01 mg toabout 10 mg. The peptide may be synthetically or recombinantly produced.Immunization may be repeated as necessary, until a sufficient titer ofanti-immunogen antibody or reactive CD4⁺ or CD8⁺ T cells has beenobtained.

In yet another alternative embodiment a viral vector, such as aretroviral vector, can be introduced into mammalian cells. Examples ofmammalian cells into which the retroviral vector can be introducedinclude, but are not limited to, primary mammalian cultures orcontinuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC#CRL 1573), B cell, dendritic or monocytic cell cultures. The means bywhich the vector carrying the gene may be introduced into a cellincludes, but is not limited to, microinjection, electroporation,transfection or transfection using DEAE dextran, lipofection, calciumphosphate or other procedures known to one skilled in the art (Sambrooket al. (eds) (1989) in “Molecular Cloning. A Laboratory Manual”, ColdSpring Harbor Press, Plainview, N.Y.). The mammalian cells expressingthe Class II restricted melanoma antigen can be administered to mammalsand serve as a vaccine or immunogen. Examples of how the cellsexpressing Class II restricted melanoma antigens can be administeredinclude, but is not limited to, subcutaneous, intravenous,intraperitoneal or intralesional. In a preferred embodiment, the nucleicacid sequence corresponding to Class II restricted peptides or modifiedtyrosinase peptides is inserted into expression vector and introducedinto the mammalian cells. By way of example, the peptides that may beused include, but are not limited to, QNILLSNAPLGPQFP (Ty 56-70) (SEQ IDNO: 1), NILLSNAPLGPQFP (Ty 57-70) (SEQ ID NO: 2), DYSYLQDSDPDSFQD (Ty448-462) (SEQ ID NO: 6), YSYLQDSDPDSFQD (Ty 449-462) (SEQ ID NO: 13),SYLQDSDPDSFQD (Ty 450-462) (SEQ ID NO: 14), QNILLSNAPVGPQFP (Ty 56-70,L65→V) (SEQ ID NO: 3), QNILLSNVPVGPQFP (Ty 56-70, A63→V and L65→V) (SEQID NO: 4), QNILLSNVPLGPQFP (Ty 56-70, A63→V) (SEQ ID NO: 5)DYSYLQDSDPDSSQD (F460→S) (SEQ ID NO: 7), DQSYLQDSDPDSFQD (Ty 448-462,Y449→Q) (SEQ ID NO: 8), DFSYLQDSDPDSFQD (Ty 448-462, Y449→F) (SEQ ID NO:9), DYSFLQDSDPDSFQD (Ty 448-462Y451→F) (SEQ ID NO: 10), DYSYLQDSVPDSFQD(Ty 448-462D456→V) (SEQ ID NO: 11), and SYLQDSVPDSFQD (Ty450-462,D456→V).(SEQ ID NO: 12) or analogs thereof. Nucleic acid sequencesencoding tyrosinase peptides having the binding core sequences providedmay also be used. Conventional methods would be used to evaluate theimmune response of the patient to determine the efficiency of thevaccine.

In yet another embodiment of this invention, the Class II restrictedtyrosinase protein, peptides or modified peptides or analogs thereof maybe exposed to dendritic cells cultured in vitro. The cultured dendriticcells provide a means of producing CD4⁺ T cell dependent antigenscomprised of dendritic cell modified antigen or dendritic cells pulsedwith antigen, in which the protein antigen is processed and expressed onthe antigen activated dendritic cell. The antigen activated dendriticcells or processed dendritic cell antigens may be used as immunogens forvaccines or for the treatment of melanoma. Alternatively the dendriticcells may present peptide antigens which have been pulsed on externally.The dendritic cells should be exposed to antigen for sufficient time toallow the antigens to bind directly to their surface MHC Class IImolecules, or to be internalized and presented on the dendritic cellssurface. The resulting dendritic cells or the dendritic cell processedantigens can than be administered to an individual in need of therapy.Such methods are described in Steinman et al. (WO93/208185) and inBanchereau et al. (EPO Application 0563485A1) which are incorporatedherein by reference. Monocytes, B cells, or Langerhans cells may besubstituted for dendritic cells.

In yet another embodiment of this invention CD4⁺ T cells isolated fromindividuals can be exposed to the Class II restricted melanoma antigenin vitro and then administered to a patient in need of such treatment ina therapeutically effective amount. Examples of where CD4⁺T-lymphocytescan be isolated, include but are not limited to, peripheral blood cellslymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes (TIL).Such lymphocytes can be isolated from the individual to be treated orfrom a donor by methods known in the art and cultured in vitro(Kawakami, Y. et al. (1989) J. Immunol. 142: 2453-3461). TheCD4⁺T-lymphocytes are cultured by methods known in the art. Thelymphocytes are exposed to peptide or protein antigen for part or all ofthe culture duration, in the presence of antigen presenting cells. In apreferred embodiment the CD4⁺lymphocytes are exposed to the Class IIrestricted tyrosinase peptides or any tyrosinase sequence having thecore peptide sequences described herein. By way of example, aconcentration of about 1 to about 200 micrograms (ug)/ml peptides per10⁷ cells for all or part of the duration of lymphocyte culture may beused. After being sensitized to the peptide the T-lymphocytes areadministered to the mammal in need of such treatment. Examples of howthese sensitized CD4⁺ T cells can be administered to the mammal includebut are not limited to, intravenously, intraperitoneally orintralesionally. Parameters that may be assessed to determine theefficacy of these sensitized T-lymphocytes include, but are not limitedto, production of immune cells in the mammal being treated or tumorregression. Conventional methods are used to assess these parameters.Such treatment can be given in conjunction with cytokines or genemodified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3:75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73). Byway of example the MHC Class II restricted melanoma antigen may beadministered in conjunction with GM-CSF to enhance uptake byprofessional antigen presenting cells in vivo.

In yet another alternative embodiment Class II restricted melanomaantigens may be linked with MHC Class II molecules and administeredeither prophylactically or therapeutically to mammals. By way of examplethe tyrosinase peptides or modified peptides described herein may becoupled with an MHC Class II molecule. Such coupling may be covalent,chemical, or genetic. By way of example, tyrosinase peptides may begenetically linked to the Class II β chain, HLA-DRB1*0401, by a flexiblepeptide linker which allows the peptide to lie in the binding groove forrecognition by T cells (Kozono, H et al (1994) Nature 369, 151-154,herein incorporated by reference). According to the promiscuous anddegenerate nature of peptide binding to MHC Class II molecules, otherDRB1 chains may be used as well (Sinigaglia, F. et al (1995) J. Exp.Med. 181, 449-451). Such single-chain Class II-MHC-peptide constructsmay be used as vaccines, or may be used to raise reactive CD4⁺ T cellsin vitro.

In yet another alternative embodiment, the aforementioned compositionscan be used to prepare antibodies to the Class II restricted melanomaantigens. The antibodies can be used directly as anti-melanomatherapeutic agents or as diagnostic reagents. Further, the antibodiescan be made even more compatible with the host system by generating“humanized” chimeric antibodies (Morrison J. 1985, Science 229:1202 andOi, et al. Biotechniques (1986) 4:214). Such antibodies can be generatedby conventional methods.

In yet another embodiment of this invention, multivalent vaccinesagainst one or more melanoma antigens are provided. Such multivalentvaccines may comprise at least one of the Class II restricted melanomaantigens described herein, preferably the tyrosinase immunogenicpeptides disclosed herein or combinations thereof, combined with otherknown melanoma antigens or peptides derived from these antigens. By wayof example, MHC Class II restricted tyrosinase peptides may be combinedin a vaccine with MHC Class I restricted peptides derived fromtyrosinase or other known melanoma associated proteins to create amultivalent vaccine capable of stimulating helper and cytotoxic immunecells. Examples of known melanoma antigens include, but are not limitedto, MART-1, gp100, gp75, MAGE-1 and MAGE-3 or immunogeneic peptidesderived from these proteins.

In another embodiment, a method is provided for identifying the presenceof Class II restricted antigenic proteins or epitopes of proteins, or ofidentifying new proteins encoding Class II restricted tumor associatedantigens, such as, but not limited to, melanoma antigens. By way ofexample, the genes or nucleic acid sequences encoding Class I restrictedmelanoma antigens can be screened for the presence of a Class IIrestricted antigenic portions or epitopes of the protein encoded bythese genes. In this embodiment, the method may comprise the steps of:(a) exposing a candidate antigen to antigen presenting cells (APC) for aperiod of time sufficient to allow the APC to take up and process theantigen; (b) incubating the APC of step (a) with CD4⁺T-lymphocytes; and(c) screening for recognition of the APC by the CD4⁺ T cells (seeExample 1).

In step (a) the candidate antigen may be presented to the APC by eitherstably on transiently expressing the gene for the candidate antigen in aeukaryotic or prokaryotic expression system. The antigen may then bepresented to the APC as crude lysates of the cells expressing thecandidate antigen or as purified protein products from the candidateantigen expressing cells. Alternatively a plurality of peptides based onthe candidate protein amino acid sequence or based on a truncatedprotein sequence derived from experiments with serial truncations of thecandidate gene may be exposed or incubated with the antigen presentingcell. It is preferred that peptides of about 15 to 20 amino acids beused.

Examples of APC that may be used in step (a) include, but are notlimited to, antigen presenting cells such as EBV transformed B celllines (Topalian et al. (1994) Int. J. Cancer 58: 69-79), monocytes anddendritic cells. Examples of how to assess recognition by the CD4⁺ Tcells incubated with the APC in step (c) include, but are not limitedto, ⁵¹CR release cytotoxicity assays (Cerundolo, V. et al. (1990) Nature345: 449-452.), cytokine secretion assays such as γ-IFN, GM-CSF or TNFsecretion. (Schwartzentruber, D. et al., (1991) J. of Immunology146:3674-3681), or proliferation assays.

Examples of proteins that may be screened for Class II restrictedmelanoma antigens includes, but are not limited to MART-1 (Kawakami, etal. (1994) Proc. Natl. Acad. Sci. 91:3575-3579), p15 (Robbins, P. F. etal (1995) J. Immunol. 154:5944-5950), MAGE-1 (VanderBruggen, Science254: 1643-1647), gp100 (Kawakami, et al. (1994) 91:6458-6462), gp75(Wang, R-F et al (1995) J. Exp. Med. 181: 799-804), and MAGE-3 (Gaugler,et al. (1994), J. Exp. Med. 179:921-930); all herein incorporated byreference).

Alternatively, this method can be used to clone and identify new geneshaving CD4 recognized tumor antigens. By way of example, a cellexpressing an unidentified tumor antigen would be assessed for CD4⁺ Tcell recognition by pulsing lysates of that cell onto antigen presentingcells (EBV-cells, monocytes, dendritic cells, etc.), and measuringcytokine secretion by T cells during coincubation. A DNA library fromthe tumor or other stimulatory cell would be expressed in a prokaryoticor eukaryotic host cell and screened according to the methods outlinedabove.

Also intended to be encompassed by this invention are Class IIrestricted tumor associated antigens, such as melanoma antigens obtainedby these methods.

Veterinary uses are also intended to be encompassed by the compositionsand therapeutic applications described herein.

All books, articles, and patents referenced herein are incorporated byreference. The following examples illustrate various aspects of theinvention and in no way intended to limit the scope thereof.

EXAMPLE I Human CD4⁺ Cells Recognize a Shared Melanoma Antigen Encodedby the Tyrosinase Gene Materials and Methods

Lymphocyte Cultures and Clones.

TIL were cultured from enzymatically digested single cell suspensions ofsolid metastatic melanoma lesions as previously described (Topalian, S.L., et al. (1987) J. Immunol. Methods. 102, 127-141), in the presence ofrecombinant interleukin-2 (rIL-2; 6000 IU/ml (Chiron Corporation,Emeryville, Calif.)), Lymphocyte cultures were passaged in bulk for 4 to6 weeks, and then CD4⁺ and CD8⁺ TIL subsets were purified by positiveselection on tissue culture flasks carrying covalently bound anti-CD4 oranti-CD8 monoclonal antibodies (mAb) (Applied Immune Sciences, Menlo,Park, Calif.) (Morecki, S., et al. (1990) J. Biol. Resp. Modif. 9,463-474). Uncloned CD4⁺ TIL were tested in bioassays after 45-70 days ofculture. CD4⁺ T cell clones were established from a 35-day bulk TIL 1088culture which was 52% CD4⁺. CD4⁺ T cells were selected and then clonedby limiting dilution in microtiter plates, in the presence of 600 IU/mlIL-2, pooled allogeneic PBL from 3 donor's (total 3×10⁴ cells/well, 3000rad; 1 rad=0.01Gy), and autologous Epstein Barr Virus (EBV)-B cells(1088-EBV 1×10⁴ cells/well, 10,000 rad) pulsed with a freeze/thaw lysateof autologous tumor (1088-mel), Clones were restimulated weekly withallogeneic peripheral blood lymphocytes (PBL), 1088-EBV, and whole1088-mel cells (1×10³ cells/well, 30,000 rad). Clones used for bioassayswere grown from 0.3 or 1 cell/well dilutions, and tested after 58-155days of culture.

B Cell Lines.

EBV-transformed B cell lines were established from the PBL of melanomapatients using standard techniques (Current Protocols In Immunology,Coligan, J. E. et al. (eds) Wiley and Sons, N.Y., N.Y. 7.22.1-7.22.3(1994) herein incorporated by reference). These lines were maintained assuspension cultures in RPMI+10% FCS.

Tumors and Normal Melanocytes.

Melanoma cultures were established from fresh or cryopreserved singlecell suspensions of metastatic lesions and maintained as adherentmonolayers in RPMI+10% FCS, as described (Topalian, S. L., et al. (1989)J. Immunol. 142, 3714-3725). Tumor clones were established by limitingdilution in 96-well flat bottom plates (Costar).

The normal melanocyte cultures FM 707, FM 708, FM 902, FM 906 and FM907, generated from neonatal foreskin, were a generous gift of Dr.Meenhard Herlyn (Wistar Institute, Philadelphia, Pa.; Herlyn, M., et al.(1985) Cancer Res. 45, 5670-5676). Cultures were maintained inMelanocyte Growth Medium (MGM, Clonetics, San Diego, Calif.), a basalserum-free medium supplemented with recombinant basic fibroblast growthfactor (1 ng/ml), insulin (5 ug/ml), hydrocortisone (0.5 ug/ml), PMA (10ng/ml), bovine pituitary extract, gentamicin (50 ug/ml), andamphotericin B (50 ng/ml). Cells were removed from this medium forseveral days prior to bioassays.

The cultured colon carcinoma CY13 was a gift of Dr. J. Yannelli (NIH,Bethesda, Md.). Colon carcinomas WiDr, LoVo, SW480; breast carcinomasZR-75-1 and MCF7; and Ewing's sarcomas 6647, RD-ES, and TC-71 were allobtained from the American Type Culture Collection (ATCC, Rockville,Md.) and maintained in RPMI+10% FCS. All cultures tested negative formycoplasma contamination.

Fresh tumor specimens were prepared from enzymatically dispersed singlecell suspensions of solid tumors, including melanomas, colon carcinomas,sarcomas, and lymphomas. They were cryopreserved in 90% FCS+10% DMSO,and were rapidly thawed for immediate use on the day of bioassay.

Antigen Presentation to T Cells by EBV-Transformed B Cells

Optimization of a bioassay for tumor-reactive CD4⁺ T cells, using EBV-Bcells as antigen presenting cells for lysates of whole tumor cells, hasbeen described (Topalian, S. L., et al. (1994) Int. J. Cancer. 58:69-79). Assay medium consisted of RPMI+10% AB serum with IL-2 120 IU/ml.Briefly, washed EBV-B cells were cultured at 7-10×10⁵ cells/ml. Tumorantigen was added to B cells in the form of cell lysates: concentratedpreparations of washed whole tumor cells were subjected to 3 cycles ofrapid freezing and thawing, and cell fragments were added to B cells at7-10×10⁵ cell equivalents/ml. Tumor-pulsed B cell cultures weremaintained at 37° C. for 20-24 h, then TIL were added at 4-10×10⁵cells/ml. Cultures were established in flat-bottom 96-well plates (220ul/well), 48-well plates (550 ul/well), or 24-well plates (1100 ul/well)depending on the numbers of cells available. As a positive control, TILwere also cultured in plates coated with anti-CD3 mAb (OKT3; OrthoPharmaceuticals, Raritan, N.J.). Cultures were maintained for anadditional 20-24 hours (h), then supernatants were harvested bycentrifugation and stored at −80° C. until assayed for the presence ofcytokines. Secreted cytokines were measured with ELISA kits purchasedfrom R+D Systems (Minneapolis, Minn.) for granulocyte-macrophage colonystimulating factor (GM-CSF, detectable concentrations 8-500 pg/ml),tumor necrosis factor alpha (TNF-α, 15-1000 pg/ml), IL-4 (31-2000 pg/ml)and IL-6 (3-300 pg/ml). GM-CSF assays were calibrated with internationalreference standard 88/646 (NCI-FCRDC, Frederick, Md.). Interferon gamma(IFN-γ) was measured with an ELISA developed with reagents fromBiosource International (Camarillo, Calif.) (20-10,000 pg/ml).

The response of TIL to tumor stimulation was considered to besignificant when cytokine secretion in response to tumor-pulsed EBV-Bcells exceeded the response to EBV-B cells alone by ≦3.0-fold.

Antibody Blocking Studies.

To inhibit TIL recognition of tumor-pulsed EBV-B cells, stimulator cellswere cultured with preservative-free mAb for 30 min at room temperaturebefore adding TIL, and then cultures were maintained for 24 h at 37° C.in the continued presence of mAb 23 ug/ml. Antibodies directed againstHLA determinants included W6/32 (against HLA-A,B,C; IgG_(2a); Sera-Lab,Sussex, England), IVA12 (HLA-DR, DP, DQ; IgG), L243 (HLA-DR; IgG_(2a)),Genox 3.53 and G2b.2 (HLA-DQw1;IgG, and IgG_(2a), respectively), andIVD12 (HLA-DQw3; IgG) (all purified from ATCC hybridoma supernatants).

Transfection of COS-7 Cells.

Genes cloned into the expression vectors pcDNA3 (Invitrogen, San Diego,Calif.) or pCEV27 (Miki, T., et al. (1991) Proc. Natl. Acad. Sci. USA88, 5167-5171) were transiently transfected into the monkey kidney COS-7cell line (a gift of Dr. W. Leonard, NIH) using the DEAE dextran method(Seed, B., & Aruffo, A. (1987) Proc. Natl. Acad. Sci. USA 84,3365-3369). EBV-B cells were pulsed with lysates of transfected COS-7cells for recognition by T cells. The tyrosinase gene was isolated froma cDNA library from the cultured melanoma line 1290A-mel, and itsidentity was confirmed by partial DNA sequencing which gave a sequenceidentical to that published by Bouchard (Bouchard, B., et al. (1989) J.Exp. Med. 169, 2029-2042). The tyrosinase (1-3) (comprising exons 1through 3) gene, also isolated from the 1290A-mel library, lacks thefourth and fifth exons and encodes a truncated product. The geneencoding tyrosinase related protein (gp75) was isolated by screening acDNA library from 501-mel with a probe constructed based on thepublished gene sequence (Vijayasaradhi, S., et al. (1990) J. Exp. Med.171, 1375-1380).

Results

CD4⁺ T cells were purified by positive selection from a heterogeneouspopulation of bulk cultured TIL derived from a metastatic melanomalesion from patient 1088. Selected cultures were >95% CD4⁺. Preliminaryexperiments indicated that these CD4⁺ T cells secreted cytokinesspecifically when cocultivated with autologous EBV-B cells (1088-EBV)which had been pulsed with lysates of autologous melanoma cells(1088-mel). TIL secreted large quantities of GM-CSF, and much smallerquantities of TNF-α, IL-4, and IFN-γ in response to autologous tumor;specific IL-6 secretion was not observed. Thus, GM-CSF secretion wasmonitored as a measure of T cell recognition in subsequent assays. In 11separate experiments, CD4⁺ TIL stimulated with tumor-pulsed EBV-B cellssecreted 8- to 138-fold more GM-CSF (median 46-fold) than TIL stimulatedwith EBV-B cells in the absence of tumor. As shown in FIG. 1, GM-CSFsecretion could be abrogated by blocking with the anti-HLA-DR mAb L243(96% inhibition), suggesting that TIL reactivity was HLA-DR restricted.Significant blocking of cytokine secretion was also observed by theanti-class II framework mAb IVA12 (71% inhibition), but not byisotype-matched mAb directed against a monomorphic MHC class Ideterminant or against two HLA-DQ determinants (HLA type of patient1088: HLA-DR 4, 17; HLA-DQw2, 3; HLA-DRw52, 53). Conversely, in the sameexperiment, cytokine secretion by purified CD8⁺ TIL 1088 in response towhole autologous melanoma cells (MHC class I⁺, class II⁻) was inhibitedby the anti-class I mAb W6/32, but not by L243 or IVA12 (data notshown). This experiment was repeated once with similar results. CD8⁺ TIL1088 capable of recognizing whole 1088-mel cells failed to react to1088-EBV cells pulsed with tumor lysate. These data indicate that CD4⁺TIL 1088 recognize autologous tumor antigen presented by EBV-B cells ina specific, MHC class II-restricted manner.

A variety of allogeneic tumors and normal tissues were screened for thepresence of Ag recognized by CD4⁺ TIL 1088. A representative experimentis shown in FIG. 2, in which lysates of the autologous cultured melanomaline as well as all 7 allogeneic melanoma cultures tested werestimulatory. Stimulation indices [SI=GM-CSF secretion by (TIL+EBV-B+celllysate)/(TIL+EBV-B)] ranged from 4.2 for 938-mel to 104.8 for 553-mel.TIL alone secreted 22 pg/ml GM-CSF, and when cocultivated with unpulsed1088-EBV secreted 101 pg/ml, as compared to 10,175 pg/ml secreted in thepresence of 1088-EBV pulsed with 1088-mel lysate. In separateexperiments, a total of 18 fresh or cultured melanomas were screened forrecognition, of which 12 were positive (67%), 4 were negative, and 2equivocal on repeat experiments. Although tumor 624-mel was recognizedby TIL 1088 on 5 separate occasions (SI=8.0 to 24.8), only 4 of 6 tumorclones from 624-mel were recognized. Taken together, these resultssuggest that the Ag recognized by CD4⁺ TIL 1088 are broadly but notuniversally expressed in melanoma lesions; alternatively, recognitionmay reflect relative degrees of antigen expression and the sensitivityof our detection system. Of note, TIL recognized autologous freshmelanoma cells (SI=19.0 and 13.1 in two experiments) as well as tumorcultured from the same lesion (1088-mel), from passages 6 through 48.Thus, the recognized antigen were present in vivo and were not afunction of culture artifact; their expression was conserved throughalmost one year of continuous in vitro culture.

Although TIL recognized a number of allogeneic melanomas on repeatassays, they consistently failed to react with normal cells ofnonmelanocyte lineage derived from the same patients. For instance, CD4⁺TIL 1088 responded to 1088-EBV pulsed with 1088-mel lysate but not tothese same EBV-B cells pulsed with 1088-EBV lysate (SI=35.7 and 1.4,respectively). This experiment was repeated twice with similar results.Also, TIL secreted significant amounts of GM-CSF in response to a lysateof fresh 501B melanoma cells, but not to lysates of EBV-B cells orcultured fibroblasts derived from the same patient (SI=13.4, 2.2, and1.5 respectively).

TIL were tested for recognition of a variety of nonmelanoma tumors, bothfresh and cultured. As represented in FIG. 3, TIL failed to recognizelysates from 14 tumors of various histologic types, including coloncarcinomas, breast carcinomas, lymphomas, and sarcomas. TIL also failedto react with two Ewing's sarcomas, which share a neuroectodermalembryonic origin with melanomas and were recognized by melanoma-specificCD8⁺ T cells in a previous study (Shamamian, P., et al. (1994) CancerImmunol. Immunother. 39:73-83). However, CD4⁺ TIL 1088 did recognize allfour normal melanocyte lines assayed, on repeated occasions. Measuredlevels of GM-CSF secretion approached those observed in response to1088-mel. These results suggested that the Ag recognized by CD4⁺ TIL1088 might be specific for the melanocytic lineage.

Two other melanoma patients whose CD4⁺ TILs recognized lysates ofautologous melanoma cells presented by autologous or HLA-matched EBV-Bcells had previously been identified (Topalian, S. et al., (1994) Int.J. Cancer 58:69-79) by our laboratory. These TILs appeared to be MHCclass II-restricted and to recognize antigens unique to autologousmelanoma cells, since they failed to react with 15 allogeneic melanomasincluding 1088-mel or with normal cells including cultured melanocytes.These results suggest the existence of multiple class II-restrictedmelanoma determinants which are differentially and specificallyrecognized by CD4⁺ T cells from these three patients.

TIL cultured in bulk under the conditions described have been shown tobe oligoclonal, but not monoclonal cell populations (Belldegrun, A., etal. (1989) J. Immunol. 142, 4520-4526; Topalian, S. L., et al. (1990) J.Immunol. 144, 4487-4495; Nishimura, M. I., et al. (1993) J. Cell.Biochem. 17D, 110. (Abstr.)). To determine whether multiple sharedantigens were being recognized by CD4⁺ TIL 1088, CD4⁺ T cell clones wereraised from these TIL and assayed for target recognition. As shown inTable 1, 4 clones recognized the autologous melanoma as well as multipleallogeneic melanomas and all three normal melanocyte lines tested (FM902, 906, 907). The target recognition profiles of all 4 clones wereremarkably similar and antibody blocking studies suggested that all wereHLA-DR restricted. For 2 clones, HLA-DR restriction was confirmed byusing allogeneic EBV-B cell lines or macrophages as APC for tumorantigen; only antigen presenting cells sharing the HLA-DR4 molecule werestimulatory. Thus, a single antigenic protein seemed to be present inall of the melanomas recognized by these CD4⁺ T cell clones, and wasshared by normal melanocytes. Further experiments with these and 7additional CD4⁺ TIL 1088 clones revealed a homogeneous recognitionprofile, suggesting the presence of an immunodominant epitope in the1088 system.

CD4⁺ TIL 1088 clones were assessed for recognition ofmelanoma-associated gene products expressed by 1088-mel on Northernblotting and known to contain commonly expressed CD8 epitopes which canbe recognized by CTL derived from melanoma patients. The genes encodingthe tyrosinase, MART-1, and gp100 proteins (Brichard, V., et al. (1993)J. Exp. Med. 178, 489-495; Kawakami, Y., et al. (1994) Proc. Natl. Acad.Sci. USA. 91, 3515-3519; Bakker, A. B. H., et al. (1994) J. Exp. Med.179, 1005-1009; Kawakami, Y., et al. (1994) Proc. Natl. Acad. Sci. USA91, 6458-6462) were cloned into plasmid vectors and transientlyexpressed in monkey kidney COS-7 cells. Lysates of transfected COS-7cells were pulsed onto 1088-EBV and used to stimulate cytokine secretionfrom CD4⁺ T cell clones. Table 2 shows that all 6 T-cell clones testedsecreted significant amounts of GM-CSF in response to lysates oftyrosinase-transfected COS-7 cells as well as to lysates from 1088-melcells. Transfection of COS-7 cells with a truncated tyrosinase gene, orgenes encoding the gp75 tyrosinase-related protein, β-galactosidase, orHLA-A2.1 did not confer recognition. As a control, CD8⁺ T cells frompatient 1088 failed to react with any of these stimulator cells. Ofnote, uncloned CD4⁺ T cells did not react as strongly as the clones withthe tyrosinase gene product. This may reflect the presence of additionalepitopes recognized by CD4⁺ TIL 1088 subpopulations not represented bythese clones, as also suggested by some discrepancies in the recognitionof allogeneic melanomas by the bulk-cultured TIL compared to the T cellclones (compare FIG. 2 to Table 1). In three additional experiments,CD4⁺ T cell clones specifically recognized the products of tyrosinasegenes isolated from two different patients' melanomas and expressed intwo different plasmids, while failing to react with MART-1 or gp100 (notshown). Taken together with previous demonstrations of CD8⁺ T cellreactivity in melanoma patients against HLA-A2 and HLA-A24 restrictedepitopes encoded by the tyrosinase gene (Brichard, et al. (1993) J. Exp.Med. 178: 489-495; Robbins, P. F. et al. (1994) Cancer Research 54:3124-3126), these findings show that a single gene product, tyrosinase,contains epitopes recognized by both CD4⁺ and CD8⁺ T lymphocytes.Furthermore, the fact that the CD4⁺ TIL react to lysates of melanomas aswell as normal melanocytes suggests that the recognized epitope isnonmutated.

The importance of CD4⁺ T cells in the priming and effector phases of theantitumor immune response has been shown in animal models (Greenberg, P.D., et al. (1985) J. Exp. Med. 161, 1122-1134; Kern, D. E., et al.(1986) J. Immunol. 136, 4303-4310; Ostrand-Rosenberg, S., Roby, C. A., &Clements, V. K. (1991) J. Immunol. 147, 2419-2422; Dranoff, G., et al.(1993) Proc. Natl. Acad. Sci. USA 90, 3539-3543). Although experimentalimmunization strategies for patients with melanoma and other cancerscurrently emphasize shared class I restricted tumor antigens recognizedby CD8⁺ T cells, immunization against both class I and class IIrestricted epitopes may increase the effectiveness of these approaches.

TABLE 1 CD4⁺ T cell clones recognize a shared antigen expressed onautologous and allogeneic melanomas and normal melanocytes GM-CSFsecretion, † pg/ml per 24 hr (SI‡) Stimulator* NT9 1D6 1B7 1E7 1088-mel14,581 (504) 27,992 (384) 21,497 (169) 13,767 (101) 1011-mel 13,626(471) 18,937 (260) 15,882 (125) 9,292 (68) 553-mel 2,856 (99) 11,067(153) 9,357 (74) 4,777 (36) 697-mel 2,451 (86) 8,887 (123) 6,497 (52)3,382 (26) 526-mel 168 (7) 2,117 (30) 1,400 (12) 196 (2) 624-mel 44 (3)1,537 (22) 1,187 (10) 98 (2) 1087-mel 37 (2) 806 (12) 675 (6) 53 (1)1290B-mel <8 (1) 168 (3) 120 (2) 33 (1) 938-mel <8 (1) 50 (2) 32 (1) <8(1) 1102-mel <8 (1) <8 (1) <8 (1) <8 (1) 586-mel <8 (1) 55 (2) 18 (1) <8(1) 537-mel <8 (1) 34 (1) <8 (1) <8 (1) 501A-mel <8 (1) 24 (1) <8 (1) <8(1) 677-mel <8 (1) 22 (1) <8 (1) <8 (1) CY13 <8 (1) <8 (1) <8 (1) <8 (1)1088-EBV <8 (1) <8 (1) <8 (1) <8 (1) FM 902 5,831 (202) 12,667 (175)10,517 (83) 6,557 (49) FM 906 11,376 (393) 17,767 (244) 16,307 (128) NTFM 907 9,401 (325) 17,382 (239) 15,202 (120) NT NT, not tested. *Celllysates of cultured lines listed were incubated with 1088-EBV cells for20 hr. In the same experiment, CD8⁺ TIL 1088 failed to react with thesestimulators (data not shown). † Net secretion = (secretion by TILs withEBV-B plus cell lysate)−(secretion by TILs with EBV-B). All cells wereat 4 × 10⁵ per ml in microtiter plates. ‡Stimulation index. GM-CSFsecretion by TIL with EBV-B in the absence of cell lysate ranged from 29to 138 pg/ml.

TABLE 2 CD4⁺ T cell clones recognize a product of the tyrosinase geneGM-CSF secretion, ‡pg/ml per 24 hr Transfected CD4⁺ CD8⁺ Stimulator*gene† bulk TILs bulk TILs NT9 1D6 1B7 1E7 2G2 2F9 1088-mel None 6169 5632,904 58,515 43,550 17,563 11,702 38,295 COS-7 Tyrosinase 123 <8 2,8047,015 9,150 2,163 12,202 39,995 COS-7 Tyrosinase (1–3) 12 <8 <8 14 <8 <8<8 <8 COS-7 Tyrosinase- <8 <8 <8 <8 46 <8 <8 37 related gp75 COS-7β-Galactosidase 8 <8 <8 16 36 16 <8 <8 COS-7 HLA-A2.1 8 <8 <8 22 <8 <8<8 12 *Cell lysates were cocultivated with 1088-EBV for 20 hr. †Geneswere expressed in the plasmid vector pcDNA3 except for thetyrosinase-related gp75 gene, which was in pCEV27. ‡Net secretion isdefined in footnote † to Table 1. Values to TIL with EBV-B ranged from10 to 237 pg/ml.

EXAMPLE II MHC Class II Restricted Tyrosinase Peptides And ModificationsThereof Materials and Methods

Peptide Synthesis. Peptides were synthesized by a solid phase methodusing a peptide synthesizer, and their molecular weights confirmed bymass spectrometry. HA 307-319 and MT(65) 3-13, with high affinities forHLA-DRB1*0401 and B1*0301, respectively, were synthesized for use asinhibitors of tyrosinase peptide binding (Sette, A. et al (1993) J.Immunol. 151, 3163-3170; Sidney J. et al (1992) J. Immunol. 149,2634-2640, herein incorporated by reference).

Tumor Cell Lines And Other Cell Lines. Same as in Example 1

Isolation of CD4⁺ TIL 1088. Same as in Example 1.

Assessment of Antigen Recognition by CD4⁺TIL 1088. EBV-B cells wereincubated overnight in the presence of peptides, up to 100 μMconcentrations. CD4⁺ T cells were added for an additional 24 hours, andcytokine secretion measured to assess T cell stimulation (see Example1).

Results. Two tyrosinase peptides recognized by bulk CD4⁺ TIL1088 havebeen identified: Ty 56-70 and Ty 448-462 (FIG. 7). This was done byscreening overlapping 15-mers based on a tyrosinase sequence derivedfrom melamona cell line 501-mel, (this sequence is the same as GenBankJ03581 sequence but with an R instead of Q at amino acid position 402).These peptides are nonmutated, since their amino acid sequences areidentical to a tyrosinase sequence derived from normal human melanocytes(Kwon et. al., PNAS (1987) 84: 7473-7477; GenBank Accession NumberJ03581).

CD4⁺ T cells from patient 1088 had been shown to recognize lysates ofautologous and some allogeneic melanoma cell lines expressingtyrosinase, and recognition (measured via cytokine secretion) wasHLA-DR-restricted (Example 1; FIGS. 1 and 2). Patient 1088 washeterozygous for DR, expressing HLA-DRB1*0301 and B1*0401. A peptidebinding HLA-DRB1*0401 with high affinity, HA 307-319, effectivelyblocked binding of both the Ty 56-70 and Ty 448-462 peptides to thepresenting EBV-B cells derived from patient 1088, leading to decreased Tcell recognition (see FIG. 4). A peptide with high affinity forHLA-DRB1*0301, MT(65)3-13, failed to inhibit T cell recognition. NeitherHA 307-319 nor MT(65) 3-13 affected T cell recognition of a melanomalysate, which is internalized by B cells and processed prior topresentation (Topalian Int. J. Cancer, 1994 58: 69-79). TheHLA-restriction of Ty 56-70 and Ty 448-462 was confirmed asHLA-DRB1*0401 by using B cell lines of various HLA genotypes as antigenpresenting cells (See FIG. 5).

In order to make more immunogenic peptides for induction of a CD4⁺ Tcell response, a variety of peptide epitopes were synthesized in whichat least one amino acid position was changed based on the binding motifsof peptides presented by HLA-DRB*0401 (Sette, A. et al (1993) J.Immunol. 151, 3163-3170; Rammensee, H. G. (1995) Immunogenetics 41,178-228, herein incorporated by reference). The P1 anchor position of Ty56-70 was hypothesized to be I58, L59, or L60 and the P6 anchor to beA63, P64 or L65 (FIG. 6). To confirm this, peptides with amino acidsubstitutions at these positions (Q=unfavorable substitution, F orV=favorable) and serially truncated peptides were tested for T cellrecognition. The results shown in FIG. 6 suggest that I58 and A63 arethe P1 and P6 anchors, respectively, of Ty 56-70 (FIG. 7). In addition,two modified peptides (Ty 56-70, A63→V and Ty 56-70, L65→V) seemed toevoke an enhanced T cell response compared to the unmodified Ty 56-70.This was confirmed by titrating the T cell response to these peptides(FIG. 8).

In repeated experiments, Ty 448-462 was specifically recognized by CD4⁺T cells from patient 1088. By testing modified peptides with eitherunfavorable (Q) or favorable (F) amino acid substitutions at potentialP1 anchor positions (FIG. 9), and by testing a series of truncatedpeptides, Y451 was identified as the P1 anchor. At this position, theunfavorable amino acid substitution abolished T cell recognition, whilethe favorable substitution restored it. In addition, it was found thatthe truncated Ty 449-462 and 450-462 peptides were more stimulatory forT cells than the parent peptide. This was confirmed in a subsequenttitration experiment (see FIG. 10).

The P6 anchor position for Ty 448-462 was presumed to be D456, based onidentifying Y451 as the P1 anchor. D (aspartic acid) is not an optimalresidue at this position, and a valine substitution led to markedlyenhanced T cell recognition (see FIG. 11). When two favorablemodifications of Ty 448-462 were combined in a single modified peptide(Ty 450-462, D456-V), recognition was enhanced even more (see FIG. 12).

In summary, both Ty 56-70 and Ty 448-462 are restricted byHLA-DRB1*0401. This MHC molecule is expressed by approximately 15% ofthe North American Caucasian population. The anchor positions in Ty56-70 and Ty 448-462 have been identified (see FIG. 7), andsubstitutions of amino acids at these positions have created modifiedpeptides with enhanced T cell stimulatory properties.

The utility of these peptides in the prophylaxis and/or therapy ofmelanoma may not be limited to patients expressing the Class II MHCmolecule DRB1*0401, as Class II-restricted peptides are often capable ofbinding to more than one Class II molecule (Chicz, R. M. et al (1993) J.Exp. Med. 178, 27-47; Malcherek, G. et al (1995) J. Exp. Med. 181,527-536).

Although the present invention has been described in some detail by wayof illustration, and examples for purposes of clarification andunderstanding it will be obvious that certain changes may be made withinthe scope of the appended claims. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the claims.

1. A purified or isolated nucleic acid molecule encoding an MHC Class IIimmunogenic portion of Tyrosinase (SEQ ID NO: 39), wherein theimmunogenic portion consists of about 9 to about 34 amino acids andcomprises at least 9 contiguous amino acids from (i) amino acids 56-70of SEQ ID NO: 39 or (ii) amino acids 448-462 of SEQ ID NO:
 39. 2. Apurified or isolated nucleic acid molecule encoding an MHC Class IIimmunogenic portion of Tyrosinase (SEQ ID NO: 39), or a derivativethereof, wherein the immunogenic portion or derivative thereof consistsof about 9 to about 34 amino acids and comprises X₁LLX₂NX₃X₄LX₅ (SEQ IDNO: 40) wherein: X₁ is any hydrophobic amino acid; X₂ is any hydrophobicamino acid; aspartic acid, or glutamic acid; X₃ is any hydrophobic orhydroxyl amino acid; X₄ is any polar, charged or aliphatic amino acid;and X₅ is any polar or aliphatic amino acid.
 3. The purified or isolatednucleic acid molecule of claim 1, further encoding a MHC Class IImolecule that is linked to the MHC Class II immunogenic portion.
 4. Thepurified or isolated nucleic acid molecule of claim 2, further encodinga MHC Class II molecule that is linked to the immunogenic portion orderivative thereof.
 5. A recombinant expression vector comprising atleast one nucleic acid molecule of claim
 1. 6. A recombinant expressionvector comprising at least one nucleic acid molecule of claim
 2. 7. Anisolated host cell containing the recombinant expression vectoraccording to claim
 5. 8. An isolated host cell containing therecombinant expression vector according to claim
 6. 9. The purified orisolated nucleic acid molecule of claim 3, wherein said MHC Class IImolecule is the β chain of the MHC Class II molecule.
 10. The purifiedor isolated nucleic acid molecule of claim 4, wherein said MHC Class IImolecule is the β chain of the MHC Class II molecule.
 11. The purifiedor isolated nucleic acid molecule of claim 1, encoding a flankingsequence at the carboxy terminus, the amino terminus, or at both terminiof the at least 9 contiguous amino acids.
 12. The purified or isolatednucleic acid molecule of claim 2, encoding a flanking sequence at thecarboxy terminus, the amino terminus, or at both termini of SEQ ID NO:40.
 13. An isolated nucleic acid molecule encoding a derivative of anisolated immunogenic peptide consisting of a portion of SEQ ID NO: 39,wherein the portion comprises (a) at least 9 amino acids from aminoacids 56-70 of SEQ ID NO: 39 with an amino acid substitution withinamino acids 56-70 of SEQ ID NO: 39 selected from the group consisting ofA63V, L65V, 158F, 158V, L60F, and L60Q, or (b) at least 9 amino acidsfrom amino acids 448-462 of SEQ ID NO: 39 with an amino acidsubstitution within amino acids 448-462 of SEQ ID NO: 39 selected fromthe group consisting of D456V, Y449F, and Y449Q, wherein the peptide is9 to 34 amino acids in length, and wherein the derivative of theisolated immunogenic peptide is presented by an MHC Class II molecule.14. The purified or isolated nucleic acid molecule of claim 1, whereinthe portion comprises at least 9 contiguous amino acids from amino acids56-70 of SEQ ID NO: 39 and comprises a substitution at amino acid 65 ofSEQ ID NO: 39 with a valine.
 15. An isolated nucleic acid moleculeencoding an immunogenic portion of Tyrosinase (SEQ ID NO: 39) consistingof about 9 to about 34 amino acids of SEQ ID NO: 39 wherein theimmunogenic portion is recognized by a CD4⁺T lymphocyte and is presentedby a Major Histocompatibility Complex (MHC) Class II molecule.
 16. Theisolated nucleic acid molecule of claim 15, further encoding an MHCClass II molecule that is linked to the immunogenic portion.
 17. Theisolated nucleic acid molecule of claim 15, further encoding a flankingsequence at the carboxy terminus, amino terminus, or at both termini ofthe immunogenic portion.
 18. A purified or isolated nucleic acidmolecule encoding an immunogenic portion of Tyrosinase (SEQ ID NO: 39)consisting of about 9 to about 34 amino acids and comprising (i) atleast 9 contiguous amino acids from amino acids 56-70 of SEQ ID NO: 39or (ii) at least 9 contiguous amino acid from amino acids 448-462 of SEQID NO: 39, wherein the immunogenic portion is recognized by a CD4⁺Tlymphocyte, which is restricted by a Major Histocompatibility Complex(MHC) Class II molecule.
 19. A purified or isolated nucleic acidmolecule encoding a derivative of an immunogenic peptide consisting of aportion of SEQ ID NO: 39 comprising at least 9 contiguous amino acidsfrom (i) amino acids 56-70 of SEQ ID NO: 39 or (ii) amino acids 448-462of SEQ ID NO: 39, wherein the immunogenic peptide is 9 to about 34 aminoacids in length and is recognized by a CD4⁺T lymphocyte, which isrestricted by a Major Histocompatibility Complex (MHC) Class IImolecule, wherein the derivative consists of a substitution at aminoacid 65 of SEQ ID NO: 39 with valine, a substitution at amino acid 63 ofSEQ ID NO: 39 with valine, or a substitution at amino acid 451 of SEQ IDNO: 39 with phenylalanine.
 20. The purified or isolated nucleic acid ofclaim 1, wherein the immunogenic peptide is SEQ ID NO: 1 or 2.